Background&#8232; Identification of genetic defects and elucidation of underlying pathways of photoreceptor degeneration constitute an essential step in developing targeted therapeutic strategies for retinal neurodegenerative diseases. Results 1. Human retinal diseases 1.1. Gene discovery: We have collected numerous small and large pedigrees for retinal and macular diseases. We are now using exome sequencing to discover new disease genes. Two or more patients/controls from more than 10 families have now been subjected to this new approach. The data is being analyzed in collaboration with Dr. Goncalo Abecasis at the University of Michigan. 1.2 We recently identified the KLHL7 gene as a cause of human autosomal dominant retinitis pigmentosa. We are performing GST pull-down/mass spectrometry experiments and yeast two-hybrid assays using human adult retinal and developing retinal yeast two-hybrid libraries. Candidate KLHL7 interacting proteins identified by GST pull-down/mass spectrometry include Myosin-9, Ncam1 and DDX1 that are currently being validated as targets for ubiquitination. 2. Animal models of human retinal disease 2.1. We are generating comparative data, including fundoscopy, ERG, histology, confocal imaging, and gene profiling of 12 retinal degeneration mutants of mice showing rapid, intermediate, and slow photoreceptor degeneration. 2.2. We have applied GFP tagging to newborn rods and BrdU birthdating to demonstrate that additional S-cones in the rd7 mouse retina originate from post-mitotic early-born photoreceptor precursors that are committed to rod fate and not by proliferation of cone photoreceptors as suggested earlier. Furthermore, we have rescued the rd7 phenotype by transgenic expression of Nr2e3 in photoreceptor precursors under control of Crx or Nrl promoters, resulting in suppression of cone genes and restoration of rod morphology/function. 2.3. We have performed detailed retinal phenotypic analysis of the rd11 and B6-JR2845 strains of mice that exhibit retinal degeneration, mapped the disease locus, and identified mutations in Lpcat1, which is critical for the production of lung surfactant phospholipids and biosynthesis of platelet-activating factor in non-inflammatory remodeling pathways. LPCAT1 is a strong candidate gene for human retinal diseases because of the importance of DPPC and lipid metabolism in photoreceptor homeostasis. 2.4. We have characterized rod and retinal gene expression in the retinal degeneration slow (rds) mouse model. We show that nearly all genes showing major (>5-fold) dysregulation display transient temporal profiles, returning to WT levels by P21. Major defects in photoreceptor structure appear to induce early protective homeostatic responses. One exception is the gene Egr-1, that is upregulated 10-fold at P21, in association with microglia activation and migration into the outer retina. This response likely represents a neuroprotective immune mechanism that contributes to the characteristically slow degeneration of the rds model. 2.5. Mutations in the centrosomal-cilia protein CEP290 lead to distinct clinical manifestations. We observed that the domain deleted in rd16 mice directly interacts with another ciliopathy protein, MKKS/BBS6. Human disease-associated MKKS/BBS6 mutations disrupt this interaction. In zebrafish embryos, combined sub-minimal knockdown of mkks and cep290 produces sensory defects Deficits in sensory cells in mice with triple allelic combinations of Cep290rd16 and Mkks-ko are less prominent than those of either single homozygous genotype. In addition, Cep290rd16/rd16;Mkksko/ko mice also demonstrate lesser functional or morphological defects and improved ciliogenesis. We suggest that the linkage between CEP290- and MKKS/BBS6-complexes is critical for cilia biogenesis and integrity and that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a novel approach for treating human ciliopathies by targeting common interacting molecules. We have now generated Cep290-knockout and genetrap mouse lines. Cep290 null allele results in early lethality around weaning, with hydrocephalus, cerebellar developmental abnormalities, and rapid retinal degeneration. CEP290 N-terminal fragment (gene trap allele) acts as a dominant negative allele, which is rescued by loss of MKKS. To model human NPHP6/NPHP5 phenotypes, we generated mice with all-cone retinas and Nphp6 dysfunction and identified an extreme dissociation of cone function and structure in the rd16;Nrl-/- retina, like that in the human ciliopathies, as well as salient features of the cone pathogenesis. We will adopt the rd16;Nrl-/- double-mutant mouse for preclinical trials in LCA with Cep290 mutations, using cone function as the metric of importance. 2.6 Reep6 knockout mice were generated to study the genes function in vivo. ERG and histological analysis show that the Reep6-/- animals develop early retinal degeneration specific to rods while cones are still functional at 8 months. This is consistent with Reep6 expression in rods and absence in cones. 2.7 Bardet-Biedl Syndrome (BBS) gene BBS9/PTHB1 encodes one of the core proteins implicated in trafficking of proteins to primary cilia. By knocking down bbs9 in zebrafish with specific antisense morpholinos we detect developmental abnormalities including hydrocephaly, retina and brain defects consistent with the core phenotypes observed in syndromic ciliopathies. We successfully rescued the bbs9 morphant phenotype using an orthologous human BBS9 mRNA, but not with one carrying a missense mutation identified in BBS patients (see also EY000451-04). 2.9. To investigate CC2D2a role in eye development, we generated CC2D2a knockout mice. Homozygous CC2D2a-/- animals are largely embryonically lethal and may show polydactyly, dextrocardia, situs inversus, indicating defective developmental signaling possibly cilia-mediated. CC2D2a-/- animals surviving at birth are blind. Initial analysis indicates that cilia are affected in several tissues, including kidney and lung. 2.9 The Nrl-KO mouse carries a retina with predominantly S-opsin containing cones that exhibit molecular and functional characteristics of WT cones. We observed that Nrl-/- retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. By four months of age, cone degeneration stabilizes, resulting in a thinned but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic ERG. The Nrl-/- retina illustrates the long-term viability of cones in the absence of rods and may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula. 2.11. We have identified a spontaneous mouse mutant showing complete absence of rod photoreceptor, absence of cone photoreceptor maturation and congenital blindness (Scotopic and photopic ERG). This mouse model will be a useful LCA model to understand the disease mechanisms. Significance Our human genetic studies have identified a number of new disease genes (RD3, CEP290, NRL, among others). Our work in animal models has highlighted an interesting convergence on biogenesis and transport functions associated with primary cilia and revealed novel pathways associated with protein degradation and lipid metabolism that contribute to photoreceptor homeostasis. These results will have significance for treating patients with LCA and retinal degeneration.